Note: Descriptions are shown in the official language in which they were submitted.
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ADJUVANT COMPOSITIONS WITH 4-1BBL
U.S. GOVERNMENT FUNDING
100011 This work was funded in part by grants from the NIH, Kentucky Lung
Cancer
Research Program, W.M. Keck Foundation, and the Commonwealth of Kentucky
Research
Challenge Trust Fund. This invention was made with government support under
Grants R41
CA121665, R44 AI071618, and R43AI074176 awarded by the National Institutes of
Health.
The government has certain rights in the invention
CROSS REFERENCE TO RELATED APPLICATIONS
[0002] This application claims benefit of the filing date of U.S. Provisional
Application No.
61/441,392, filed February 10,2011.
FIELD OF THE INVENTION
[0003] The present invention generally relates to the surprisingly effective
synergy
observed when combining a toll-like receptor (TLR) agonist such as
monophosphoryl lipid A
(MPL) with 4-1BBL as adjuvants, and to related compositions and methods to
enhance the
immune response against antigens, and related methods of preventing and
treating diseases,
including cancer.
BACKGROUND
[0004] Therapeutic vaccines are preferred alternatives to conventional
treatments for cancer
primarily because of their safety profile and generation of long-term
immunological memory
that is critical for the control of disease recurrence, which is the main
cause of death from
cancer. Therapeutic vaccines based on tumor associated antigens (TAAs) are
particularly
attractive because of their ease of production, scale-up, storage, and
administration to a broad
patient population. The efficacy of such vaccines, however, is curtailed by
the weak
antigenic nature of self TAAs due to both central and peripheral tolerogenic
mechanisms (1,
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2). Despite continued promising results, there is a greater than two-decade
track record of
failure with cancer vaccines. Similar failures are also found in other
immunity contexts,
including vaccines against infectious diseases, self antigens, and against
haptens such as
toxins and addictive drugs.
100051 3-0-desacy1-4'- monophosphoryl lipid A (MPL), is a nontoxic version of
lipopolysaccharide approved by the FDA to be used as the adjuvant component of
a
preventive vaccine against HPV infection (5). MPL is also under investigation
as an adjuvant
in other contexts. MPL is a toll-like receptor 4 (TLR-4) agonist, like lipid
A,
lipopolysaccharide and other structurally related compounds. CpG is a
structurally dissimilar
TLR agonist. MPL primarily targets innate immunity, leading to the
recruitment, activation,
and maturation of antigen presenting cells (APCs), such as dendritic cells
(DCs) that facilitate
the generation of adaptive immune responses (6).
[0006] 4-1BBL is a member of the TNF family. 4-1BBL is a costimulatory
molecule that
targets CD8+ T cells for activation, acquisition of effector function,
survival, and long-term
memory (15-17). 4-1BBL is also able to stimulate CD4+ Treg cells to induce
tolerance
against a given antigen (U.S. Patent no. 7,745,215). 4-1BBL is expressed on
the cell surface
and has no function in soluble form. The extracellular functional domain of 4-
1BBL can be
fused to a modified form of streptavidin (SA) to generate a chimeric molecule
(SA-4-1BBL)
that exists as tetramers and oligomers owing to the structural features of SA
(12).
SUMMARY OF THE INVENTION
[0007] The present invention is drawn to the surprising discovery of the
synergy of toll-like
receptor (TLR) agonists such as monophosphoryl lipid A (MPL) and 4-1.BBL as an
adjuvant
composition to induce immune responses against a co-administered antigen. In
some
embodiments there are provided compositions comprising 4-1BBL and a TLR
agonist. In
some embodiments, the TLR agonist is a TLR4 agonist, such as
lipopolysaccharide, lipid A
or a chemical analogue, such as MPL. In some embodiments, there are provided
compositions comprising 4-1BBL and MPL. 4-1BBL may be provided in a fusion
protein,
such as a streptavidin-4-1BBL fusion protein. In some embodiments, the 4-1BBL
fusion
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protein forms higher order structures, such as trimers and aggregates, via the
streptavidin
moieties. In some embodiments, the composition further comprises a
pharmaceutically-
acceptable excipient.
[0008] In further embodiments, the composition further comprises an antigen.
The antigen
may be provided in a conjugate with 4-1BBL, such as a conjugate comprising
biotinylated
antigen complexed with SA-4-1BBL. Suitable antigens include a tumor or cancer-
associated
antigen, a self antigen, an antigen associated with an infectious agent, or a
hapten. Suitable
cancer or tumor-associate antigen include: human telomerase reverse
transcriptase (hTERT),
survivin, MAGE-1, MAGE-3, human chorionic gonadotropin, carcinoembryonic
antigen,
alpha fetoprotein, pancreatic oncofetal antigen, MUC-1, CA 125, CA 15-3, CA 19-
9, CA
549, CA 195, prostate-specific antigens, prostate-specific membrane antigen,
Her2/neu, gp-
100, mutant K-ras proteins, mutant p53, truncated epidermal growth factor
receptor, chimeric
p
protein 210 BCR-ABL, E7 protein of human papilloma virus, EBNA3 protein of
Epstein-Barr
virus, cTAGE-1 and variants, BLA or globotriaosylceramide (Pk antigen), human
T-cell
leukemia virus-associated cell membrane antigens (HTLV-MA), Thymocyte surface
antigen
JL1, Adult T cell leukemia associated, human retrovirus associated antigen
(ATLA),
Anaplastic lymphoma kinase (ALK), fusion proteins (NPM/ALK and variants),
Common
acute lymphoblastic leukemia antigen (CALLA), Immunoglobulin Id, Type II
glycoproteins
(such as HM1.24, KW-2, KW-4, KW-5, KW-12), Oncofetal antigen immature laminin
receptor protein (OFA-iLRP), and EBV proteins (e.g., LMP2A).
[0009] Antigens associated with an infectious agent include those from HIV,
influenza,
malaria, tuberculosis, staphylococcus, and streptococcus.
[0010] Also provided are methods of inducing an immune response against an
antigen in a
subject, comprising administering to the subject (a) the antigen, (b) a TLR
agonist, and (c) 4-
1BBL, such as SA-4-1BBL. Suitable TLR agonists include TLR4 agonists, such as
lipopolysaccharide, lipid A and chemical analogues, such as MPL. A second
and/or
subsequent administrations may also be performed. Again, the antigen may be
provided in a
conjugate with 4-1BBL. In practicing the methods, any two or more of the
antigen, TLR
agonist (such as MPL), and 4-1BBL may be administered as part of the same
composition, or
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may be administered simultaneously or sequentially in separate compositions by
the same or
different routes of administration.
[00111 Also provided are methods of treating a tumor or a cancer in a subject,
comprising
administering to the subject (a) an antigen associated with the tumor or
cancer, (b) a TLR
agonist, such as MPL, and (c) 4-1BBL, such as SA-4-1BBL. Suitable TLR agonists
include
TLR4 agonists, such as lipopolysaccharide, lipid A and chemical analogues,
such as MPL.
A second and/or subsequent administrations may also be performed with the same
or
different antigen. Again, the antigen may be provided in a conjugate with 4-
1BBL. Again,
any two or more of the antigen, TLR agonist (such as MPL), and 4-1BBL may be
administered as part of the same composition, or may be administered
simultaneously or
sequentially in separate compositions by the same or different routes of
administration.
[00121 In any of the embodiments described herein, a further immune
costimulatory
molecule, such as OX-40L/CD137L, can be used in addition to the 4-1BBL The
further
immune costimulatory molecule (e.g., OX-40L/CD137L) can be provided in a
fusion protein
comprising avidin or streptavidin. In practicing the methods, the further
immune
costimulatory molecule may be administered as part of the same composition as
any one or
more of the antigen, TLR agonist (such as MPL), and 4-1BBL, or may be
administered
simultaneously or sequentially in separate compositions by the same or
different routes of
administration.
BRIEF DESCRIPTION OF THE DRAWINGS
[00131 Figure 1. A single vaccination with the SA-4-1BBL/MPL adjuvant system
results
in the eradication of established TC-1 tumor in mice. (A) C57BL/6 mice were
challenged
subcutaneously with lx105 live TC-1 cells and left unvaccinated (PBS) or
vaccinated once
subcutaneously on day 6 post-tumor challenge with E7 (50 g) mixed with
control SA
protein (10 pig) or SA-4-1BBL (25 tag), MPL (25 g), or the combination of
both agents (25
g/agent). The log-rank test and Kaplan-Meier method were used for analyses. *P
< 0.05 as
compared to all the other groups, but SA-4-1BBL that was not significant (ns).
(B) Data
from (A) are presented for individual animals in each group.
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[0014] Figure 2. Vaccination with the SA-4-1BBL/MPL adjuvant system induces
strong
anti-tumor CD8+ T cell effector and memory responses that correlate with
vaccine efficacy.
Long-term (> 90 days) surviving mice were boosted with the same indicated
vaccine
formulations used for primary vaccination. Lymph node cells were harvested 7
days later
and assessed for E749-57 peptide-specific CD8 T cells expressing (A)
intracellular IFN-y
mono, (B) IFN-yTNF-a double, and (C) IFN-yTNF-cdL-2 triple cytokines. (D)
Splenocytes
from the same groups were phenotyped to test the percentage of effector memory
CD44h1CD62LI0wCD8+ T cells. Data for each panel are representative of two
independent
experiments that include 3-4 mice per group. P values were as shown and
calculated using
one way ANOVA and Tukey HSD test (ns = not significant).
[0015] Figure 3. Vaccination with the SA-4-1BBL/MPL adjuvant system results in
an
increase in the intratumoral Teff/Treg cell ratio. Mice bearing TC-1 tumor (-3-
4 mm in
diameter; n = 4 per group) were vaccinated subcutaneously with E7 protein (50
pig) alone or
with SA-4-1BBL (25 MPL (25 jig), or a combination of both agents (25
jig/agent). One
week post-vaccination, tumors were harvested and stained for intratumoral CD8f
T cells and
CD4+Foxp3+ Treg cells followed by analysis using confocal microscopy. (A)
Confocal
pictures of tumor sections showing CD4 Foxp3+Treg cells (top panel; bright
cells) stained
with anti-CD4 antibody, anti-Foxp3 antibody, and Hoechst, and CD8+ T cells
(bottom panel;
bright spots) stained with anti-CD8 antibody and Hoechst. (B) Quantitative
analysis of
intratumoral CD4+Foxp3+ Treg cells, (C) CD8+ T cells, and (D) CD8+ Teff/Treg
cell ratio. P
values were as shown and calculated using one way ANOVA and Tukey HSD test (ns
= not
significant).
[0016] Figure 4. Therapeutic efficacy of SA-4-1BBL/MPL adjuvant system
requires CD8+
T cells while Treg cells compromise the efficacy of MPL monotherapy. CD84- T
cells and
Treg cells were depleted using antibodies against CD8 and CD4 molecules,
respectively, one
day before vaccination with E7 TAA and the indicated adjuvant system using the
TC-1
established tumor model. Data for PBS, E7+MPL, and E7+MPL+SA-4-1BBL groups
were
taken from Fig. 1.
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[0017] Figure 5. Vaccination with the SA-4-1BBL/MPL adjuvant system generates
potent
therapeutic response in the 3LL lung metastasis model. Mice (n = 4-5/group)
were
challenged with 2x105 live 3LL cells by intravenous tail injection and
vaccinated once
subcutaneously on day 6 or twice on days 6 and 13 post-tumor challenge with
survivin (SVN)
(50 pig) alone or antigen with SA-4-1BBL (25 MPL (25 jug), or a combination
of both
agents (25 ig/agent). (A) Lungs were harvested 27 days post tumor challenge
and assessed
for tumor growth by weight and macroscopic presence of tumor nodules. (B)
Intracellular
IFN-y response of CD8+ T cells was assessed after with phorbol 12-myristate 13-
acetate
(PMA) and ionomycin stimulation of lymphocytes harvested from mice in (A). (C)
Lungs
harvested from mice with two vaccinations are assessed as in (A). P values
were as shown
and calculated using one-way ANOVA and Post Hoc LSD test (ns = not
significant).
[0018] Figure 6. Vaccination with the SA-4-1BBL/MPL adjuvant system does not
promote autoimmunity. Sera were harvested from mice challenged with 3LL tumor
cells
shown in Fig. 5A and TC-1 tumor cells in Fig. lA at the experimental end
points and tested
for the presence of autoantibodies against single stranded DNA (ssDNA) in
ELISA. Serum
pooled from a minimum of 3 naive and 3 lupus mice were used as negative and
positive
controls, respectively.
[0019] Figure 7. Vaccination with the SA-4-1BBL/MPL adjuvant system induces
robust
primary CD8+ T cell effector functions. (A) Tumor draining lymphocytes from
mice (n = 4)
shown in Fig. 3 were stimulated with E749-57 peptide and analyzed for CD8+ T
cell
intracellular IFN-y expression. (B) Splenocytes from mice in (A) were
stimulated with E749-
57 peptide and IL-2 for 5 days and used as effectors against TC-1 tumors. 3LL
tumor cells
were used as irrelevant targets. The data is representative of 3 independent
experiments. P
values were as shown and calculated using one-way ANOVA and Tukey HSD test (ns
= not
significant).
[0020] Figure 8 depicts the amino acid sequences of representative fusion
proteins, (A)
core streptavidin (CSA) with the extracellular domain of murine 4-1BBL and (B)
core
streptavidin (CSA) with the extracellular domain of human 4-1BBL. The core
streptavidin
sequence is underlined. (C) Fusion of hexahistidine-streptavidin-ALA3 tag onto
murine 4-
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1BBL (6XHis-SA-ALA3-m4-1BBL). The 6XHis-SA-ALA3 tag is underlined. (D) Fusion
of
Flag streptavidin and A1a3 tags onto human CD137 ligand (1XFlag-SA-A1a3-
CD137L). The
1XFlag-SA-A1a3 tag is underlined.
DETAILED DESCRIPTION OF THE INVENTION
[0021] For the purposes of the present application, the following terms have
these
definitions:
[0022] As used herein "a" or "an" means one or more, unless specifically
indicated to mean
only one.
[0023] "Administration" as used herein encompasses all suitable means of
providing a
substance to a patient. Common routes include oral, sublingual, transmucosal,
transdermal,
rectal, vaginal, subcutaneous, intramuscular, intravenous, intra-arterial,
intrathecal, via
catheter, via implant etc. In some embodiments, a composition is administered
near or
directly to the tumor, such as by direct injection into the tumor or injection
into the blood
such as when the tumor is a tumor of the blood.
[0024] An "adjuvant" increases the immune response against an antigen with
which it is
presented. Adjuvants are known in the art and include aluminium hydroxide,
aluminium
phosphate, monophosphoryl lipid A, oils, cytokines, and the like. Oil-in-water
and water-in-
oil emulsions also are used as adjuvants. Antigen carriers such as virosomes
and immune-
stimulating complexes (e.g., ISCOM and ISCOMATRIX) also may result in enhanced
presentation of antigens. (See Table 1 of Leroux-Roels, Vaccine 28S: C25-C36
(2010))
[0025] "Antigen" is used herein without limitation. Antigens include proteins,
lipids,
sugars, nucleic acids, chemical moieties, and other moieties that induce an
immune response.
Antigens include proteins, which may or may not be modified, such as by
glycosylation or
methylation, that are cyclized or bound to lipids, for example. Antigens
associated with an
infectious agent or disease include antigens that are part of the infectious
agent, such as
envelope proteins, capsid proteins, surface proteins, toxins, cell walls,
antigenic lipids, and
the like. Other antigens may be expressed only in the presence of the host.
Other suitable
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antigens may, in some embodiments, include antigens of the host, including
those that are
induced, modified or otherwise overexpressed as a marker of infection or
disease. All such
antigens that are derived from, or associated with, an infectious agent, an
infection, a
condition or disease, are suitable for use in the present invention. Also
suitable for use as an
"antigen" in accordance with the present invention are peptides comprising
antigenic portions
of full-length proteins, such as peptides comprising a portion of a protein
that induces an
immune response, such as an immunogenic epitope. For example, suitable
antigens may
include synthetic peptides that induce an immune response.
[0026] In some embodiments, the antigen is an "allergen." An allergen is an
antigen which
induces the symptoms of allergic disease, such as IgE antibodies against the
allergen, MAST
cell degranulation and/or the release of histamine in the presence of the
allergen. In such an
embodiment, the goal may be less concerned with developing an immune response
to the
antigen, per se, but rather in changing the nature of the immune response. For
example,
inducing an immune response to the allergen that stimulates the production of
IgG, instead of
IgE antibodies. This may be achieved through the induction of a TH1 over TH2
response.
[0027] "Excipients" includes vehicles, carriers, diluents, pH adjusting and/or
buffering
agents, tonicity adjusting agents, stabilizers, wetting agents, binders, and
the like.
Pharmaceutically acceptable excipients are well known in the art. For example,
alum
(hydrated potassium aluminium sulfate, KA1 (SO4)2.1 2H20), is commonly used in
vaccine
formulations as an excipient to bind and stabilize biological molecules and as
an adjuvant.
[0028] "Immune co-stimulatory polypeptide" means a polypeptide molecule that
increases
an individual's immune response against a pathogen (including an infectious
agent) or tumor.
In addition to 4-1BBL, OX4OL is an exemplary immune co-stimulatory polypetide
that can
be used in the compositions and methods described herein.
[0029] "Immune cell" as used herein includes any cell that is involved in the
generation,
regulation or effect of the acquired or innate immune system. Immune cells
include T cells
such as CD4+ cells, CD8+ cells and various other T cell subsets, B cells,
natural killer cells,
macrophages, monocytes, dendritic cells, and neutrophils.
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[0030] "Patient" or "subject" as used herein includes any mammal. In some
embodiments,
the patient is human. A person of ordinary skill in the art will recognize
that particular
immune co-stimulatory molecules, signaling molecules, cell markers, cell
types, infectious
agents etc., discussed with reference to one species, may have corresponding
analogues in
different species, and that such analogues, and their use in corresponding and
related species,
are encompassed by the present invention.
[0031] "Toll-like receptor agonist" (or TLR) as used herein are molecules
which bind to
and activate toll-like receptors. Agonists and antagonists of different TLRs
are known. Lipid
A is a lipopolysaccharide found in the outer membrane of gram negative
bacteria that acts as
a potent TLR4 agonist. MPL is a chemical analogue of lipid A that also acts as
a TLR4
agonist.
[0032] "Tumor" as used herein includes solid and non solid tumors (such as
leukemia); and
different stages of tumor development from pre-cancerous lesions and benign
tumors, to
cancerous, malignant and metastatic tumors.
[0033] A "vaccine" describes a preparation designed to induce an immune
response against
an antigen. A vaccine may be therapeutic, given during treatment to boost the
immune
response or drive the response in a specific direction, or it may be
prophylactic or
preventative, given prior to or shortly after exposure to a disease. A vaccine
does not have to
induce a fully protective response that prevents or eradicates all evidence of
disease, as not all
vaccines produce an immune response in all people, and the strength and nature
of the
immune response varies between people. A vaccine may be both therapeutic and
prophylactic at the same time, in treating an existing condition and
preventing future
recurrences.
Toll-like receptor agonists.
[0034] The toll-like receptors are a family of proteins involved in innate
immunity.
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Toll-like receptors and agonists
TLR Localization Ligand Origin of the
Ligand
TLR1 Plasma membrane Triacyl lipoprotein Bacteria
TLR2 Plasma membrane Lipoprotein Bacteria, viruses,
parasites, self
TLR3 Endolysosome dsRNA Virus
TLR4 Plasma membrane LPS Bacteria, viruses,
self
TLR5 Plasma membrane Flagellin Bacteria
TLR6 Plasma membrane Diacyl lipoprotein Bacteria,
viruses
TLR7 Endolysosome ssRNA Virus, bacteria,
self
(human TLR8)
TLR9 Endolysosome CpG-DNA Virus, bacteria,
protozoa, self
TLR10 Endolysosome Unknown Unknown
TLR11 Plasma membrane Profilin-like molecule Protozoa
(See Table 1 of Takeuchi & Akira, Cell 140: 805-820 (2010)).
TLR agonists in clinical use or development include
(a) MPL, a TLR4 agonist and a chemical analogue of lipidA/LPS. MPL may also be
used in
combination with alum or QS21 (a saponin);
(b) synthetic derivatives of dsRNA (TLR3);
(c) S. typhimurium flagellin (TLR5);
(d) imiquidazoquinoline derivatives (TLR7 and/or 8); and
(e) Immunostimultory sequences such as synthetic phophorothioate-linked DNA
oligonucleotides with optimized CpG motifs (TLR9)
See Table 1 of Leroux-Roels, Vaccine 28S: C25-C36 (2010); See also Table 1 of
Coffman et
al., Immunity 33: 492-503 (2010).
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4-1BBL
[0035] Immune co-stimulatory molecules are involved in the natural interaction
between
naive T cells and antigen presenting cells, which results in their reciprocal
activation and
prompts the expression of various cell surface ligands and receptors, and
soluble proteins that
contribute to the initiation, maintenance, and long-term memory of the immune
response. At
least three signals are required for the initial activation of naive T cells.
Signal 1 is generated
by interactions between a T cell receptor (TCR) and a nominal peptide
presented by major
histocompatibility complex (MHC) molecules on the surface of professional APC,
such as
dendrtic cells (DC). Signal 2 can be mediated by several different molecules
and is important
to a sustained immune response. Signal 3 is transduced via cytokines
elaborated by activated
T cells and APC and is important to the maintenance of effector immune
responses.
[0036] 4-1BBL (also known as 4-BB-L, 4-BB ligand, TNFSF9, ILA ligand) is a
type II
protein expressed on activated B cells, macrophages, and DC two to three days
following
activation. 4-1BB/4-1BBL interactions also transduce Signal 2 to CD8+ T cells
in a CD28-
independent manner and stimulate them to produce cytokines, expand, and
acquire effector
functions.
[0037] 4-1BBL contains 254 amino acids (26624 Da). See Alderson et al. Eur J
Immunol.
1994 Sep;24(9):2219-27. The full amino acid sequence of human 4-1BBL can be
found
under accession no. P41273 in the Swiss-Prot database. 4-1BBL is a type II
glycoprotein
with residues 1-28 forming a potential cytoplasmic domain, residues 29-49
forming a single
predicted transmembrane domain, residues 50-254 forming a potential
extraceulluar domain,
and residues 35-41 representing a poly-Leu stretch. The nucleotide sequence in
humans
encoding the 4-1BBL can be found in GenBank accession no. NM_003811. Residues
50-254
of 4-1BBL or fragments thereof that can bind to its cognate receptor 4-1BB,
can be linked or
expressed as a fusion with a binding pair member for use in accordance with
the present
invention. U.S. Patent No. 7,598,345.
[0038] Unless specified herein as "full-length," reference herein to 4-1BBL
polypeptide
encompasses the full-length polypeptide as well as fragments or portions
thereof that exhibit
immune co-stimulatory function, including, but not limited to those fragments
and portions
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specifically identified below. Thus, for example, reference to a 4-1BBL
polypeptide
connotes a polypeptide comprising a fragment or portion of full-length 4-1BBL
that exhibits
immune co-stimulatory function, such as the extracellular domain of 4-1BBL or
the full-
length 4-1BBL protein. In some embodiments, the immune co-stimulatory
polypeptide does
not comprise the transmembrane domain of 4-1BBL. In some embodiments, the
immune co-
stimulatory polypeptide comprises at least the extracellular domain of 4-1BBL,
or a receptor-
binding fragment thereof. "IMMUNOSTIMULATORY COMPOSITIONS AND
METHODS" describes suitable fusion proteins between 4-1BBL and streptavidin.
Additional immune costimulatory polypeptides
[00391 In any of the embodiments described herein, a further immune
costimulatory
molecule, can be included in the composition in addition to the 4-1BBL. The
further immune
costimulatory molecule can be provided in a fusion protein comprising avidin
or streptavidin.
[0040] Immune co-stimulatory polypeptides include, without limitation, LIGHT,
CD80
(B7-1), CD86 (B7-2), ICOS, ICOSL (including B7h, B7-H2, B7RP-1, GL-50 and
LICOS),
CD94 (KP43), CD4OL (CD154), ICAM-1 (CD54), ICAM-2, ICAM-3, SLAM (CD150), HAS
(CD24), 4-1BB (CDw137), OX4OL, CD28, CD40 (BP50), CD25 (IL-2R a), Lymphotoxin
(LTa or LT(3), TNF, Fas-L, GITR (activation-inducible TNRF), GITR Ligand,
CD1la (a L
integrin), CD1 lb (am integrin), L-selectin (CD62L), CD69 (very early
activation antigen),
CD70 (CD27L), PD-L1, PD-L2, B7-H3, B7-H4, OX4OL, 4-1BBL, CD27L, CD3OL, LIGHT,
BAFF, and APRIL. See, e.g., Watts & DeBenedette, 1999, Curr. Opin. Irnmunol.,
11:286-93.
[0041] In some embodiments, the compositions and methods of the invention
further
comprise OX4OL. OX4OL is expressed by dendritic cells and other APC and binds
to 0X40
which is present on activated T cells. OX4OL contains 183 amino acids (21950
Da). See
Miura etal. Mol. Cell. Biol. 11:1313-1325 (1991). The full amino acid sequence
of OX4OL
can be found under accession no. P23510 in the Swiss-Prot database. OX4OL is a
type II
glycoprotein with a cytoplasmic domain at residues 1-23, a transmembrane
domain at
residues 24-50 and an extracellular domain at residues 51-183. The nucleotide
sequence of
OX4OL is 3510 bp, with the coding sequence being 157-708 (see Genbank
accession no.
NM 003326.2). Residues 51-183, or fragments thereof of OX4OL that can bind to
its
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cognate receptor 0X40, can be linked or expressed as a C-terminal fusion to a
binding pair
member for use in accordance with the present invention. U.S. Patent No.
7,598,345 for
"IMMUNOSTIMULATORY COMPOSITIONS AND METHODS" describes suitable fusion
proteins between OX4OL and streptavidin.
Fusion proteins
[0042] In exemplary embodiments, the 4-1BBL is comprised in a fusion protein
with
streptavidin (SA) or avidin, or fragments thereof which retain substantial
properties of the
full-length protein. Such fragments include "core streptavidin" ("CSA"), a
truncated version
of the full-length streptavidin polypeptide which may include streptavidin
residues 13-138,
14-138, 13-139 or 14-139. The nucleic acid sequences encoding streptavidin and
avidin and
the streptavidin and avidin amino acid sequences can be found, for example, in
GenBank
Accession Nos. X65082; X03591; NM 205320; X05343; Z21611; and Z21554. When
another immune costimulatory peptide is used, such as OX-40L, it also may be
provided in a
fusion protein with SA or avidin.
[0043] SA and CSA are able to aggregate into trimers and higher order
structures; thus, SA-
4-1BBL conjugates (or other immune costimulatory conjugates, such as SA-0X-40L
conjugates) may form trimers and higher order structures that may be necessary
for immune
costimulation. Another property of SA, CSA and avidin is binding to biotin,
and fragments
with at least 50% or more of the binding affinity of native SA or avidin,
respectively, also
may be used. The biotin-binding property may be used to target or localize 4-
1BBL (or other
immune costimulatory molecule, such as OX-40L) to a target site or surface, or
to attach
another molecule, such as an antigen.
Antigens & Infectious Agents
[0044] The methods and compositions described herein are useful for generating
or
enhancing an immune response against any antigen or infectious agent,
including TAAs,
antigens associated with an infectious agent, and an infectious agent itself.
An antigen
associated with the targeted tumor or infectious agent (or the infectious
agent itself) may be
presented to immune cells, thereby generating or enhancing an immune response.
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1. TAAs
[0045] In one embodiment, the antigen is a tumor associated antigen (TAA), and
the
compositions and methods provide cancer immunotherapy effective to generate or
enhance a
patient's immune response against a tumor. In accordance with this embodiment,
the
methods and compositions may reduce tumor size and/or inhibit the growth of
tumor cells.
[0046] Representative tumor cells which may be targeted include, without
limitation,
carcinomas, which may be derived from any of various body organs including
lung, liver,
breast, bladder, stomach, colon, pancreas, skin, and the like. Carcinomas may
include
adenocarcinoma, which develop in an organ or gland, and squamous cell
carcinoma, which
originate in the squamous epithelium. Other cancers that can be treated
include sarcomas,
such as osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage),
leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle),
mesothelial
sarcoma or mesothelioma (membranous lining of body cavities), fibrosarcoma
(fibrous
tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma
(adipose
tissue), glioma or astrocytoma (neurogenic connective tissue found in the
brain),
myxosarcoma (primitive embryonic connective tissue), an esenchymous or mixed
mesodermal tumor (mixed connective tissue types). In addition myelomas,
leukemias, and
lymphomas are also susceptible to treatment.
[0047] A number of TAAs associated with specific tumor types have been
identified.
These include human telomerase reverse transcriptase (hTERT), survivin, MAGE-
1, MAGE-
3, human chorionic gonadotropin, carcinoembryonic antigen, alpha fetoprotein,
pancreatic
oncofetal antigen, MUC-1, CA 125, CA 15-3, CA 19-9, CA 549, CA 195, prostate-
specific
antigens; prostate-specific membrane antigen, Her2/neu, gp-100, mutant K-ras
proteins,
mutant p53, truncated epidermal growth factor receptor, chimeric protein
P210BCR-ABL; E7
protein of human papilloma virus, and EBNA3 protein of Epstein-Barr virus. Any
of these
antigens, antigenic fragments thereof, and mixtures of antigens and/or
fragments can be used
in accordance with the compositions and methods described herein to generate
or enhance a
patient's anti-tumor immune response. Table 5 lists some exemplary TAAs and
diseases
associated with such TAAs.
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Table 5.
Antigen Diseases
cTAGE-1 and variants Cutaneous T cell lymphoma
BLA or globotriaosylceramide Burkitt's lyrnhoma
(Pk antigen)
human T-cell leukemia virus-associated Adult T-cell leukemia lymphoma (ATL)
cell membrane antigens (HTLV-MA)
Thymocyte surface antigen JL1 Majority of acute leukemias
Adult T cell leukemia associated, human Adult T cell leukemia
retrovirus associated antigen (ATLA)
Epstein-Barr virus (EPV) antigens Burkitt's lymphoma, Hodgkin's disease
Anaplastic lymphoma kinase (ALK), CD30+ anaplastic large cell lymphoma
fusion proteins (NPM/ALK and variants) (ALCL)
Common acute lymphoblastic leukemia Most acute lymphoblastic leukemias
antigen (CALLA)
Immunoglobulin Id; Type II Lymphoproliferative diseases
glycoproteins (e.g., HM1.24; KW-2,
KW-4, KW-5, KW-12); Oncofetal
antigen immature laminin receptor
protein (OFA-iLRP); EBV proteins (e.g.,
LMP2A)
100481 Additional human TAAs recognized by T-cells may be found in, for
example,
Novellino etal. "A listing of human tumor antigens recognized by T cells:
March 2004
update" Cancer Immunology and Immunotherapy, 54: 187-207 (2005) which is
incorporated
by reference herein. Many animal TAAs corresponding to animal corollaries of
these
diseases, and to other animal diseases, are known in the art and also included
within the scope
of the invention.
[00491 In one embodiment, the TAA is selected from the group consisting of
human
telomerase reverse transcriptase (hTERT) and survivin. hTERT is expressed in
>85% of
human cancers, while its expression is restricted in normal tissues. See,
e.g., Vonderheide et
al., Immunity 1999, 10: 673-79. Similarly, survivin, which has been identified
as an inhibitor
of apoptosis, is absent from normal tissues but expressed in most tumor types
including lung,
colon, pancreas, prostate and breast cancer. See, e.g., Ambrosini et al., Nat.
Med. 1997, 3:
917-21. Because these TAAs are expressed in the majority of cancer types and
are rare or
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absent from noonal tissues, they are attractive antigens for use in cancer
immunotherapy
methods according to the present invention.
[0050] In another embodiment, the TAA is associated with cervical cancer. The
viral
transforming proteins, E6 and E7 (also known as "early" proteins), are
consistently expressed
in cervical cancer cell lines and in FWV-associated cancers, and are
consistently expressed in
most cervical cancers. Because E6 and E7 are completely foreign viral
proteins, and may
harbor more antigenic peptides or epitopes than a mutant protein, and thus
have several
benefits as a TAA for therapeutic purposes.
[0051] TAAs may be admixed with 4-1BBL and MPL for administration. The TAA may
also be conjugated to, for example, 4-1BBL, such as through a biotin linkage.
2. Infectious Agents
[0052] Representative infectious agents against which the compositions and
methods
described herein may be applied include, without limitation, any virus,
bacteria, fungi or
protozoan. Table 6 lists examples of infectious agents.
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TABLE 6
ETIOLOGICAL GENUS ASSOCIATED
AGENT DISEASE
BACTERIAL Mycobacterium Tuberculosis
tuberculosis
Bacillus anthracis Anthrax
Staphylococcus Sepsis
aureus
Borrelia spp Lyme disease
Salmonella Typhoid
VIRAL Adenoviridae Mastadenovirus Infectious canine
hepatitis
Arenaviridae Arenavirus Lymphocytic
choriomeningitis
Caliciviridae Norovirus Norwalk virus infection
Coronaviridae Coronavirus Severe Acute
Respiratory Syndrome
Toro virus
Enterovirus Hand-foot and mouth
disease, diarrheal
disease
Filoviridae Marburgvirus Viral hemorrhagic
fevers
Ebolavirus Viral hemorrhagic
fevers
Flaviviridae Flavivirus West Nile Encephalitis
Hepacivirus Hepatitis C virus
infection
Pestivirus Bovine Virus Diarrhea,
Classical swine fever
Hepadnaviridae Orthohepadnavirus Hepatitis
Herpesviridae Simplexvirus cold sores, genital
herpes, bovine
mammillitis
Varicellovirus chickenpox, shingles,
abortion in horses,
encephalitis in cattle
Cytomegalovirus infectious
mononucleosis
Mardivirus Marek's disease
Orthomyxoviridae Influenzavirus A Influenza
Influenzavirus B Influenza
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ETIOLOGICAL GENUS ASSOCIATED
AGENT DISEASE
Papillomaviridae Papillomavirus Skin warts (including
genital warts), skin
cancer, cervical cancer
Picornaviridae Enterovirus Polio
Rhinovirus Common cold
Aphthovirus Foot-and-mouth disease
Hepatovirus Hepatitis
Poxviridae Orthopoxvirus Cowpox, vaccinia,
smallpox
Reoviridae Rotaviruses Diarrhea
Orbivirus Blue tongue disease
Retroviridae Gammaretrovirus Feline leukemia
Deltaretrovirus Bovine leukemia
Lentivirus Human
immunodeficiency, FIV,
and SIV
Rhabdoviridae Lyssavirus Rabies
Ephemerovirus Bovine ephemeral fever
Togaviridae Alphavirus Eastern and Western
equine encephalitis
PARASITIC Plasmodium Malaria
Leishmania Leishmaniasis
FUNGAL Aspergillis
Candida
Coccidia
Cryptococci
Geotricha
His toplasma
Microsporidia
Pneumocystis
[0053] Human and avian influenza, HIV, hepatitis C, tuberculosis, West Nile
Virus,
cryptococcosis (meningitis) herpes, chlamydia, and anthrax are representative
of infectious
agents. Any antigen associated with the infectious agent can be used in
accordance with the
invention. Any infectious agent may be used, such as a virus, including a
human or avian
influenza virus or HIV, or any other virus. Other infectious agents and
antigens derived
therefrom include Hepatitis A, C or E virus, Japanese encephalitis virus,
Dengue virus,
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Hantavirus, Rabies virus, and SARS coronavirus. The infectious agent may be
modified or
attenuated to reduce or eliminate its infectivity.
[0054] In accordance with one embodiment, the antigen or infectious agent is
provided in a
conjugate with 4-1BBL, such as by biotinylation of the agent and binding to
the SA moiety
on SA-4-1BBL (e.g., forming an Antigen-Biotin-SA-4-1BBL complex).
[0055] For the purpose of illustration only, this aspect is described in more
detail with
reference to influenza. Influenza is a contagious disease caused by the
influenza virus, and
affects the respiratory tract, often resulting in symptoms in the nose, throat
and lungs, as well
as fever, headache, tiredness and aches. It can also lead to complications
such as pneumonia,
bronchitis, or sinus and ear infections or exacerbate chronic conditions.
Influenza viruses are
classified as type A, B or C. Strains belonging to types A and B circulate in
the population
and are associated with most cases of human influenza. Type A influenza causes
the
overwhelming majority of public health problems in humans. In this context,
the antigen
may comprise one or more of H1 and Ni (both highly immunogenic) and/or one or
more of
nucleoprotein (NP) and matrix protein 1 (MP1) and/or matrix protein 2 (MP2)
(all highly
conserved, structural proteins). Proteins from pandemic strains such as H5,
also can be used
as antigens in accordance with the invention.
[0056] The compositions and methods described herein can be used in influenza
vaccines
that are easy to produce and manufacture quickly, whose antigenic component
can be
changed and updated based on the current health needs without difficulty, that
selectively
targets viral machinery and infected cells, and that can be administered post-
infection for a
therapeutic effect as well as pre-infection for prevention.
100571 The compositions and methods described herein are useful as vaccines
against other
infectious agents, which can be selected in an analogous manner by those
skilled in the art,
based on the antigens associated with those infectious agents. For example,
antigens
associated with HIV include HIV envelope gp120 epitopes (e.g., variable loops
such as V3),
or other HIV proteins such as Gag proteins (Pr55gag, matrix p17, capsid p24,
nucleocapsid
p7), p5; Pol (polymerase), Vif (viral infectivity factor p23); Vpr (viral
protein R p15); Rev
(regulator of viral gene expression p19); Vpu (viral protein U); Env (gp 160,
gp120, gp41);
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Tat (transcriptional activator p14); and Nef (negative effector p24). See,
e.g., Peters, 201,
Vaccine 2: 688-705; Michael, 2003, Clin. Med. 3: 269-72; Gandhi & Walker,
2002, Ann.
Rev. Med. 53: 149-72; Haseltine, 1991, FASEB 5: 2349-60. Other antigens useful
in
vaccines include capsular polysaccharides of Haemophilius influenzae type b,
capsular
polysaccharides of Neisseria meningitidis, capsular polysaccharides of
Streptococcus
pneumoniae, surface antigens of Hepatitis B, and inactivated exotoxins of
diphtheria and
tetanus toxins. These antigens can be used in accordance with the composition
and methods
described above with reference to influenza antigens.
Allergens
[0058] The term "allergen" refers to antigens associated with allergies. An
allergic response
is characterized by the release of inflammatory factors, particularly
histamine, leading to
pathologic inflammation in an individual. Allergies are, typically, also
associated with IgE
antibodies directed against the allergens. Examples of allergens include, but
are not limited
to: pollens (e.g. grass, ragweed, birch and mountain cedar); house dust and
dust mites;
mammalian epidermal allergens and animal danders; mold and fungus; insect
bodies and
insect venom; feathers; food; and drugs (e.g., penicillin). The compositions
and methods of
the invention are suitable to induce or modulate an immune response against an
allergen that
is of a different nature to a preexisting immune response. For example, an
individual
possessing a TH2 immune response against an allergen such that IgE antibodies
are produced
upon exposure to the allergen may be induced, by embodiments of the present
invention, to
produce a TH1 immune response against the allergen, that counteracts the
allergy inducing TH2
response and so alleviate allergic disease.
Compositions
100591 As set forth above, some embodiments of the invention relate to
compositions.
These include compositions comprising 4-1BBL and a toll-like receptor (TLR)
agonist, such
as MPL. 4-1BBL may be provided in a fusion protein, such as a streptavidin-4-
1BBL fusion
protein or a core streptavidin-4-1-BBL fusion protein. The compositions may
also include
pharmaceutically-acceptable excipients that are well-known in the art, and may
be prepared
and/or provided in suitable forms for storage (frozen, lyophilized, etc.)
and/or administration.
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Some excipients may also exhibit adjuvant properties. An example of such an
excipient is
alum (hydrated potassium aluminium sulfate (Potassium alum) with the formula
KA1(504)2.12H20). Alum can bind to and stabilize variant antigens, and is also
used as an
adjuvant. The 4-1BBL/TLR agonist composition is suitable for use as an
adjuvant, and
therefore may include or be administered with an antigen. The antigen may be
included in
the 4-1BBL/TLR agonist composition at the time of manufacture, or can be added
later, prior
to or upon administration. The antigen may be a separate component of the
composition, or
may be part of a conjugate that also comprises 4-1BBL, such as may be prepared
by
biotinylation of the antigen and binding to the streptavidin moiety of SA-4-
1BBL. As
discussed above, any antigen can be used, such as from cancer, tumors, and
infectious
diseases.
100601 In any of the embodiments described herein, a further immune
costimulatory
molecule, such as OX-40L/CD137L, can be included in the composition in
addition to the
4-1BBL The further immune costimulatory molecule (e.g., OX-40L/CD137L) can be
provided in a fusion protein comprising avidin or streptavidin.
Methods
100611 As set forth above, some embodiments of the invention relate to
methods. For
example, the methods described herein are useful to induce an immune response
against an
antigen, such as an antigen associated with tumors or cancers, or infectious
agents. In some
embodiments, such methods comprise administering to a subject (a) the antigen,
(b) a toll-
like receptor (TLR) agonist, such as MPL, and (c) 4-1BBL. The methods
described herein
also are useful for treating a tumor or cancer by administering to a subject
(a) the antigen,
(b) a toll-like receptor (TLR) agonist, such as MPL, and (c) 4-1BBL. In any of
these
methods, a second and/or subsequent administrations of a same or similar
agents (e.g., the
same or different antigen) may be performed. As noted above with reference to
compositions, the 4-1BBL may be provided in a fusion protein, such as a
streptavidin-
4-1BBL fusion protein or a core streptavidin-4-1-BBL fusion protein and/or the
antigen may
be provided as a separate component or in a conjugate that also comprises 4-
1BBL. Further,
in practicing the methods, any two or more of the antigen, TLR agonist, and 4-
1BBL may be
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administered as part of the same composition, or may be administered
simultaneously or
sequentially in separate compositions by the same or different routes of
administration.
[0062] In any of the embodiments described herein, a further immune
costimulatory
molecule, such as OX-40L/CD137L, can be administered in addition to the 4-1BBL
The
further immune costimulatory molecule (e.g., OX-40L/CD137L) can be provided in
a fusion
protein comprising avidin or streptavidin, and can be provided in the same
composition as the
4-1BBL or can be provided in a separate composition that is administered
simultaneously
with or sequentially (i.e., before or after) the 4-1BBL composition.
[0063] Thus, in practicing the methods described herein, one may administer
antigen, a TLR
agonist, 4-1BBL, and optional additional costimulatory molecules,
simultaneously in the
same or different compositions, or sequentially, at the same or different
sites, and at the same
or different times and in different orders.
[0064] The following examples illustrate the invention in more detail, and are
not intended
to limit the scope of the invention in any respect.
EXAMPLES
EXAMPLE 1. 4-1BBL/MPL SYNERGY
MATERIALS AND METHODS
Mice and cell lines
[0065] C57BL/6 and C57BL/6.SJL mice were bred in a barrier animal facility at
the
University of Louisville. All animals were cared for in accordance with
institutional and NIH
guidelines. TC-1 and 3LL cell lines were purchased from ATCC (Manassas, VA)
and
maintained as published (7).
Antibodies and other reagents
[0066] Fluorochrome-conjugated anti-CD8-APC-Cy7, anti-CD62L-PE, anti-CD44-APC,
anti-TNF-PE, anti-IFN--y-PE-Cy7, and anti-IL-2-PerCp-Cy5.5, and isotype
controls were
purchased from BD Bioscience, eBioscience, and BioLegend. MPL was purchased
from
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InvivoGen (San Diego, CA). The HPV16 RAHYNIVTF E7 peptide (E749-57), SA-4-
1BBL,
E7 and mouse SVN proteins were reported previously (7).
Tumor models and vaccination
[0067] C57BL/6 mice were challenged subcutaneously with lx i05 live TC-1 cells
into the
right flank. For therapy, mice were vaccinated subcutaneously on day 6 post-
tumor challenge
with various vaccine foimulations containing E7 protein (50 g) alone as
control or with SA-
4-1BBL (25 pig), MPL (25 ps), or the combination of both agents (25 g/agent).
The doses
of E7, SA-4-1BBL, and MPL used in this study were based on previously
published studies
(7). Mice were euthanized when tumor reached a size of 12 mm in diameter,
ulcerated, or
mice showed signs of discomfort. CD8+ and CD4+ T cells were depleted using
antibodies
against CD8 (clone 53.6.72) and CD4 (clone GK 1.5) at 500 g/mice via intra-
peritoneal
injection one day before vaccination
[0068] For the pulmonary tumor model, 2x105 live 3LL cells were injected
intravenously
into the tail vein of mice. Mice were vaccinated subcutaneously either once on
day 6 or twice
on days 6 and 13 post-tumor challenge with various vaccine formulations
containing survivin
(SVN) protein (50 g) alone as control or with SA-4-1BBL (25 g), MPL (25
gig), or the
combination of both agents (25 g/agent). Mice were euthanized 27 days post-
tumor
challenge for analysis of lung tumor burden as described (12, 18).
Flow cytometry and confocal microscopy
[0069] Spleens and/or tumor draining lymph nodes (TdLNs) were processed as
described
previously (7). For memory T cell typing, lymphocytes were stained with anti-
CD8-APC-
Cy7, anti-CD62L-FITC, and anti-CD44-APC antibodies. For intracellular cytokine
staining,
lymphocytes (1x106 cells/mL) were stimulated either with 10 ug/mL E749_57
peptide for 2 hrs
followed by incubation with GolgiPlug (1 1/mL, BD PharMingen) overnight or
with phorbol
12-myristate 13-acetate (PMA) (5 ng/ml, Sigma) and ionomycin (500 ng/ml,
Sigma) for 2 hrs
followed by incubation with GolgiPlug (1 1/m1) for an additional 4 hrs. Cells
were first
stained with anti-CD44-APC and anti-CD8-APC-Cy7, fixed with 4%
parafolinaldehyde, and
then stained with anti-IFN-T-PE-Cy7, anti-IL-2-Percp-Cy5.5, anti-TNF-PE, or
isotype
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controls followed by acquisition and analysis as previously reported (12).
Intratumoral CD8+
T cells and CD4+Foxp3+ Treg cells were analyzed using confocal microscopy as
described
(12).
Analysis of autoantibody to ssDNA
[00701 A single stranded DNA (ssDNA) ELISA was performed to assess the
presence of
auto-antibodies in treated mice as described (19). Briefly, ninety six titer
plates coated with 1
ps/well of heat-denatured calf thymus DNA (ssDNA, Sigma) were blocked with PBS
containing 5% BSA + 0.5% Tween 20 + 0.1% naive C57BL/6 serum. Serum dilutions
were
added to wells and incubated at 4 C overnight. Wells were washed 3 times,
incubated with
anti-mouse IgG-HRP, and absorbance was measured at 450 nm.
Cytotoxicity assay
[00711 Mice with ¨3-4 mm TC-1 tumors in diameter were vaccinated
subcutaneously with
vaccine formulations containing E7 protein (50 flg) alone or with SA-4-1BBL
(25 g), MPL
(25 tg), or the combination of both agents (25 g/agent). One week post-
vaccination,
splenocytes were cultured with 10 ps of E749-57 peptide/mL in complete MLR
medium
supplemented with 50 IU/mL of IL-2. Viable lymphocytes were harvested 5 days
later using
a Ficoll gradient and used as effectors at various ratios against TC-1 target
cells for 4 hrs as
described (41). See Figures 7A and 7B.
RESULTS
Combined use of SA-4-1BBL and MPL as the adjuvant component of E7 TAA-based
vaccine has robust efficacy in eradicating established TC-1 tumors
[00721 A single vaccination with SA-4-1BBL and E7 protein was effective in
eradicating
E7 expressing established TC-1 tumors in > 70% of mice (12). Although
impressive, the
present inventors have discovered that the therapeutic efficacy of this
vaccine can further be
improved by modifying the formulation to include MPL as a second adjuvant with
a primary
effect on innate immunity. A single subcutaneous vaccination with E7 protein
mixed with
SA-4-1BBL and MPL resulted in the eradication of established TC-1 tumors in
all mice,
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which remained tumor-free over an observation period of 90 days (Fig. 1A). It
was
completely surprising to achieve 100% effectiveness, i.e. in all mice, and for
such an
extended period.
100731 By comparison, monotherapy with SA-4-1BBL or MPL resulted in
eradication of
tumors in only 80% and 50% of mice, respectively. Mice that expired from
tumors in the
single agent groups, however, had slow kinetics of tumor progression as
compared with both
PBS and E7 protein control groups, where all mice expired from the tumor
burden within 50
days (Fig. 1B). Taken together, these data demonstrate that SA-4-1BBL/MPL as
an adjuvant
system is effective in eradicating the established TC-1 tumors with better
therapeutic efficacy
than the individual agents, and that SA-4-1BBL has better efficacy than MPL.
The therapeutic efficacy of the vaccine is associated with the synergistic
effects of SA-4-
1BBL and MPL on the generation of peripheral CD8+ T cell responses
[0074] CD8+ T cell effector and memory responses are critical to the
elimination of the
primary tumor and control of recurrences, respectively, in various tumor
settings, including
the TC-1 model (7, 10, 11, 13). The CD8+ T cell effector and long-term memory
responses
elicited by various vaccine formulations were assessed as follows. Mice that
had eradicated
the tumor in response to various vaccine formulations were boosted
subcutaneously with the
same formulations and then euthanized one week later to test the intracellular
cytokine
response of CD8+ T cells to the dominant E749-57epitope (10). Consistent with
the
therapeutic efficacy, vaccination with E7 protein and SA-4-1BBL/MPL generated
a better
antigen-specific cytokine response than single adjuvant therapy as assessed by
CD8+ T cell
expressing IL-2, IFN-y, and TNF-ot triple cytokines (Fig. 2A-C). Consistent
with the
therapeutic responses, mice vaccinated with SA-4-1BBL formulation generated
significantly
(P < 0.05) better IFN-y response than the MPL formulation (Fig. 2A). A vaccine
formulation
with SA-4-1BBL/MPL also generated the most effective CD8+ T cell memory recall
responses as compared to those including SA-4-1BBL and MPL as single adjuvants
(Fig.
2D). Collectively, these data demonstrate that SA-4-1BBL and MPL adjuvants
work in
synergy to generate potent CD8+ T cell effector and memory responses that
correlate with the
therapeutic efficacy of the vaccine against the TC-1 tumor.
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Vaccination with the SA-4-1BBL/MPL adjuvant system results in a favorable
intratumoral CD8+ Teff/Treg cell ratio
[00751 Elevated levels of intratumoral CD4 Foxp3+ Treg cells along with a
decline in CD8+
Teff cells is associated with a clinically unfavorable prognosis of cancer
patients (21, 22) and
depletion of Treg cells results in better immune efficacy of therapeutic
vaccines (23, 24).
The effect of the SA-4-1BBL/MPL adjuvant system on the status of intratumoral
Treg and
Teff cells was evaluated as follows:
[0076] Mice bearing ¨3-4 mm TC-1 tumor were vaccinated subcutaneously with
various
vaccine foi ____________________________________________________________ -
nulations. One week post-vaccination, tumors were harvested and analyzed for
the presence of intratumoral CD8+ T cells and CD4+FoxP3+ Treg cells using
confocal
microscopy. There was a significant reduction in the number of intratumoral
Treg cells in
mice vaccinated with either SA-4-1BBL as a single adjuvant or in combination
with MPL
when compared with PBS controls or E7 protein alone. (Fig. 3A, B).
Interestingly, vaccine
formulation containing MPL as a single adjuvant did not have detectable effect
on the
number of intratumoral Treg cells as compared with PBS control, and indeed
performed
worse than E7 protein alone that appreciably, but not statistically
significantly, reduced the
intratumoral number of Treg cells.
[00771 The following data show that a decrease in the number of Treg cells
caused by SA-
4-1BBL/MPL or SA-4-1BBL as monotherapy inversely correlates with the number of
intratumoral CD8+ T cells, a hallmark of a successful immunotherapeutic
approach against
cancer (25). Vaccination with SA-4-1BBL/MPL had the most pronounced effect on
the
number of intratumoral CD8+ T cells infiltration, followed by SA-4-1BBL,
whereas MPL
had a moderate effect that was similar to the E7 protein alone (Fig. 3C). This
increased
intratumoral CD8+ T cells by SA-44BBL/MPL resulted into the most favorable
intratumoral
Teff/Treg cell ratio, followed by SA-4-1BBL as monotherapy (Fig. 3D). In
marked contrast,
MPL as a single adjuvant had no effect on the intratumoral Teff/Treg cell
ratio as compared
with both PBS and E7 protein controls. Significantly (P < 0.05) better E7 TAA-
specific
CD8+ T cell IFN-7 (Figure 7A) as well as TC-1 killing (Figure 7B) responses
were observed
in mice vaccinated with both adjuvants as compared with a single adjuvant.
Consistent with
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the therapeutic efficacy and infiltration of intratumoral CD8+ T cells, SA-4-
1BBL as
monotherapy generated better CD8+ T cell IFN-y as well as killing responses
than E7 antigen
alone, whereas MPL failed to do so. Taken together, these findings demonstrate
that SA-4-
1BBL and MPL work in synergy to increase the intratumoral Teff/Treg cell ratio
that
correlates with the potent efficacy of this adjuvant system in eliminating
established tumors.
CD8+ T cells are correlated with the therapeutic efficacy of SA-4-1BBL/MPL
adjuvant
system while Treg cells are detrimental to the efficacy of MPL monotherapy
[0078] To test if a high CD8+ Teff/Treg cell ratio can serve as a predictor of
vaccine
therapeutic efficacy, we used antibodies against CD8 and CD4 molecules to
deplete CD8+
Teff and Treg cells, respectively. Mice with established TC-1 tumors were
treated with
depleting antibodies one day before vaccination with E7 protein admixed with
SA-4-
1BBL/MPL or MPL as monotherapy. As shown in Fig 4, depletion of CD8+ T cells
completely abrogated the therapeutic efficacy of SA-4-1BBL/MPL adjuvant
system, while
depletion of CD4+ T cells, including Treg cells, improved the therapeutic
efficacy of MPL
from 50 to 100%. Taken together, these data provide direct evidence for the
opposing roles
of CD8+ T and Treg cells in vaccine efficacy and point to the importance of
Teff/Treg cell
ratio as a predictor of vaccine efficacy/failure.
Vaccination with SA-4-1BBL/MPL adjuvant system and survivin eradicates
established
3LL pulmonary metastatic tumors
[0079] Efficacy of vaccination with survivin (SVN), a weak and potentially
tolerant self-
TAA, was assessed in the 3LL pulmonary metastasis model. Mice were challenged
intravenously with a lethal dose of live 3LL cells followed by subcutaneous
vaccination on
day 6 with various formulations containing SVN recombinant protein and SA-4-
1BBL and/or
MPL as adjuvants. As shown in Fig. 5A, the vaccine formulation containing both
adjuvants
had the most therapeutic efficacy over the single adjuvant composition in
controlling tumor
growth, as demonstrated both by lung weight and presence of tumor nodules.
Similar to the
TC-1 model, the vaccine foimulation containing SA-4-1BBL as sole adjuvant had
better
efficacy in controlling tumor growth than MPL as sole adjuvant, which had a
statistically
significant (P < 0.05) effect in controlling tumor growth over the controls of
PBS or
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adjuvant-free SVN. The therapeutic efficacy of SA-4-1BBL/MPL combination
therapy, or
SA-4-1BBL monotherapy, but not MPL monotherapy, correlated with significantly
(P <
0.05) higher number of CD8+ T cells expressing IFN-7 as compared with PBS and
SVN
alone controls (Fig. 5B).
[0080] Although lungs of SA-4-1BBL/MPL vaccinated mice had similar weights as
compared with lungs of naive mice, some of the lungs had microscopically
detectable tumor
nodules. We therefore, tested the efficacy of a booster injection 7 days after
the first
vaccination. As shown in Fig. 5C, boosting with SA-4-1BBL/MPL with SVN
resulted in
complete eradication of lung tumor in all mice. Booster vaccination with
single adjuvants
was also effective in eradicating and/or controlling tumor burden that reached
statistical
significance (P < 0.05) as compared with PBS and SVN alone controls.
Collectively, these
findings further confii n the utility of SA-4-1BBL/MPL as a powerful
adjuvant system to
elicit potent immune responses to a self-TAA that translates into effective
immunotherapy in
a stringent pulmonary preclinical metastasis model.
Therapeutic efficacy of the SA-4-1BBLIMPL adjuvant system is achieved in the
absence
of detectable clinical toxicity and autoimmunity
[00811 Autoimmunity is a potential setback to effective self-TAA-based
therapeutic
vaccine formulations using potent adjuvants to induce immune responses to such
antigens
(26). Given the potent therapeutic activity of the adjuvant system described
herein, we tested
serum from mice with successful immunotherapy for both the TC-1 as well as 3LL
models,
for the presence of antibodies against single stranded DNA (ssDNA) as a sign
of systemic
autoimmunity. There was lack of significant amount of auto-antibodies to ssDNA
in all the
groups tested, whereas the serum from mice with full blown lupus had high
levels of such
antibodies (Fig. 6). Importantly, signs of acute toxicity in vaccinated mice
were not detected,
including weight loss, unexpected mortality, gross anatomy, and macroscopic
analysis of
body organs, demonstrating the safety profile of this adjuvant system.
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DISCUSSION
[0082] MPL and SA-4-1BBL as the adjuvant component of an HPV E7 TAA-based
vaccine
synergized to generate a robust primary CD8+ T cell effector and long-term
memory
responses that translated into improved therapeutic efficacy in the TC-1
cervical cancer
mouse model. The therapeutic efficacy of the adjuvant system was totally
dependent on
CD8+ T cells and associated with a favorable intratumoral CD8+ Teff/CD4+Foxp3+
Treg cell
ratio. Moreover, the synergy of the adjuvant system is not limited to the
xenogenic E7 TAA.
A vaccine formulation containing the adjuvant combination with SVN, as a bona
fide self-
TAA, was equally effective in eradicating/controlling tumors in the 3LL
metastatic
pulmonary cancer model.
[0083] While not wishing to be bound by theory, it would appear that MPL in
the adjuvant
system synergizes with SA-4-1BBL for the activation of CD8+ T cells through
the activation
of DCs and antigen cross-presentation (27), resulting in the upregulation of 4-
1BB receptor
on the surface of CD8+ T cells that in turn become the direct target of SA-4-
1BBL. This
scheme is supported by our data reported herein, showing that MPL synergizes
with SA-4-
1BBL in generating robust CD8+ T cell primary and long-teini memory responses
that
translate into effective therapy in two different established tumor models, TC-
1 cervical and
3LL pulmonary carcinoma, with two different antigens, HPV E7 xenogenic and SVN
bona
fide self-TAA antigens. Consistent with this hypothesis, depletion of CD8+ T
cells one day
before vaccination completely abrogated the efficacy of the SA-4-1BBL/MPL
adjuvant
system in eradicating TC-1 tumors. In addition to its direct effect on CD8+ T
cells, SA-4-
1BBL may also augment the effect of MPL on DCs by improving their antigen
uptake and
cross-presentation. This hypothesis is supported by observations that a
subpopulation of DCs
constitutively express 4-1BB receptor (28, 29), and vaccination with SA-4-1BBL
enhances
their antigen uptake and cross-presentation (7, 12).
[0084] CD4+CD25+FoxP3+ Treg cells play a critical role in immune evasion
mechanisms
employed by acute (30) as well as chronic infections (31, 32) and cancer (2,
23, 33), and as
such serve as an important barrier for the efficacy of vaccines. Therefore,
vaccine
formulations that specifically control the number and/or function of Treg
cells while
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enhancing the number of Teff cells may have desired therapeutic efficacy in
settings of
cancer and chronic infections. Consistent with this hypothesis are studies
demonstrating that
the physical depletion of Treg cells or modulation of their regulatory
function using
antibodies to various cell surface markers have protective and therapeutic
effects against
various tumors in preclinical models (33-37). A recent study using mice
transgenically
expressing the diphtheria toxin receptor only in Treg cells demonstrated that
specific and
conditional depletion of these cells protected mice from carcinogenesis-
induced spontaneous
tumors via innate immunity and eradicated established tumors via CD8+ T cell-
and IFN-y-
dependent responses (36). Consistent with preclinical studies, Treg cells were
shown to
accumulate in various progressing cancers in patients and a high intratumoral
Teff/Treg cells
ratio is considered the hallmark of a favorable prognosis (21-23).
[0085] Important in this context are the results reported here, showing a
robust increase in
the ratio of intratumoral CD8+ Teff/Treg cells in response to vaccination with
the SA-4-
1BBLIMPL adjuvant system. Vaccination with SA-4-1BBL as monotherapy also
significantly improved the intratumoral CD8+ Teff/Treg cell ratio.
Surprisingly, MPL as
monotherapy was not only inefficient in significantly increasing the frequency
of
intratumoral CD8+ T cell infiltration, but also failed to decrease the
intratumoral number of
Treg cells, resulting in an unfavorable CD8+ Teff/Treg cell ratio. The Treg
cells played a
detrimental role in the efficacy of MPL-based vaccine since their depletion
one day before
vaccination resulted in eradication of all tumors (Fig. 4). This finding,
demonstrating that
MPL efficacy is compromised by Treg cells is significant, and provides an
important
mechanistic insight for improving therapeutic cancer vaccines.
[0086] Although MPL primarily targets cells of innate immunity, a series of
recent studies
have demonstrated that this adjuvant may also directly target cells of
adaptive immunity. The
expression of TLR-4 has been shown on CD4+ T effector and Treg cells (38, 39).
Importantly, stimulation via this receptor on CD4+ Teff cells was shown to
inhibit ERK1/2
signaling pathway, resulting in the inhibition of their function in an
experimental colitis
model (39). In marked contrast, stimulation of Treg cells with the TLR-4
agonist
lipopolysaccharide resulted in their survival, expansion, and improved
regulatory function in
vivo (38), which may account for the unfavorable intratumoral CD8+ Teff/Treg
cell ratio seen
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in the MPL monotherapy group. Although the exact mechanistic basis of the
synergistic
effect of SA-4-1BBL and MPL on the intratumoral CD8+ T/Treg cell ratio
observed in our
model is unknown and, without wishing to be bound by theory, the following may
apply:
(i) SA-4-1BBL may preferentially induce apoptosis in Treg cells as reported
for the agonists
of OX-40 pathway (40), another close member of TNFR costimulatory family; (ii)
SA-4-
1BBL may block the tumor-mediated conversion of Teff cells into induced Treg
cells and
(iii) SA-4-1BBL and MPL may both increase the intratumoral frequency of CD8+
Teff cells,
thereby favorably influencing the CD8+ Teff/Treg cell ratio. These mechanisms
are
supported by additional (unpublished) data demonstrating that SA-4-1BBL blocks
tumor- and
TGF-0-induced conversion of Teff cells into induced Treg cells through IFN-y.
The
increased expression of IFN-y in response to an SA-4-1BBL/MPL adjuvant system
is further
consistent with this theory. Although enhanced E7 TAA-specific frequency of
CD8+ T cells
expressing IFN-y was observed in the periphery of mice vaccinated with MPL as
monotherapy, this effect did not result in increased number of CD8+ T cells in
the tumor,
suggesting that these cells may not be trafficking into the tumor. In
contrast, vaccination
with the combined SA-4-1BBL/MPL adjuvant resulted in significantly higher
numbers of
CD8+ Teff cells both in the periphery and within the tumor, suggesting that
both adjuvants in
combination may affect the trafficking,/entry of CD8+ Teff into the tumor
and/or improve
their survival. Thus, and while not wanting to be bound by theory,
immunomodulation with
SA-4-1BBL is believed to block the conversion of T effector cells into T
regulatory cells,
which may be a factor for the robust therapeutic efficacy of the MPL/SA-4-1BBL
combination.
[0087] The therapeutic activity of the combined SA-4-1BBL/MPL adjuvant was
achieved
in the absence of detectable acute toxicity and chronic autoimmunity. The lack
of acute
toxicity is consistent with previously published studies demonstrating that
treatment of mice
with 4-fold higher SA-4-1BBL over the therapeutic dose used in these studies
did not result
in detectable toxicity, as assessed by systemic cytokine response, non-
specific
lymphoproliferation, altered lymphocyte trafficking, generalized lymphomegaly
and
splenomegaly, and hepatitis, all of which were observed with similar doses of
an agonistic
antibody to 4-1BB receptor (14). The safety of MPL has already been
demonstrated both in
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preclinical and clinical settings (5, 6, 20). Nevertheless, the complete
absence of acute or
chronic pathologies is surprising in view of the high potency of the adjuvant
combination.
100881 The results presented above demonstrate the robust efficacy of the SA-4-
1BBL/MPL adjuvant combination for inducing potent CD8+ Teff primary and long-
term
memory responses against TAAs and a favorable intratumoral CD8+ Teff/Treg cell
ratio that
translate into potent therapeutic efficacy in two different tumor models, and
which was
observed in the absence of detectable acute toxicity or chronic autoimmunity.
[0089] In conclusion, the combination of monophosphoryl lipid A (MPL) and 4-
1BBL is
surprisingly effective as an adjuvant. When administered with an antigen, an
MPL/4-1BBL
adjuvant combination results in an immune response that is very potent, e.g.
curing 100% of
cancers in all mice from a single immunization, a result not previously
reported. Not only is
the response potent, but also of a type that is particularly effective for
therapy and for long
teito immunity. Notably, the MPL/4-1BBL adjuvant combination stimulated the
production
of CD8+ Teti cells, and resulted in a favorable Teff/Treg ratio. The Teff/Treg
ratio is an
important predictor of long term survival from cancer. Also, the MPL/4-1BBL
adjuvant
combination resulted in the infiltration into tumors of CD8+ Teff cells,
consistent with an
active immune response against the tumor. Despite the strong and effective
immune
response, the MPL/4-1BBL adjuvant combination composition did not cause any
detectable
symptoms of acute or chronic toxicity, such as an autoimmune response.
Accordingly, a
composition comprising MPL and 4-1BBL as an adjuvant is highly effective in
inducing an
immune response against cancers and tumors, and can also be used against other
conditions,
such as treatment for, or prophylaxis against, infectious agents. Because MPL
is toll-like
receptor (TLR) agonist, the present inventor believes that other TLR agonist
adjuvants may
exhibit a synergistic effect when used with 4-1BBL as described herein.
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